JP3009458B2 - Plasmid containing a DNA sequence encoding a human placental ribonuclease inhibitor - Google Patents

Abstract

The in vitro cloning of a gene for human placental ribonuclease inhibitor may be obtained by recombinant DNA technology. The method described obtains the gene encoding human placental ribonuclease inhibitor, the expression of that product in a host cell, and the refolding and purifying of the product. A vector which includes an encoded DNA gene sequence coding for human placental ribonuclease inhibitor is also described, as well as a host that is compatible with and contains the vector. The gene sequence of the human placental ribonuclease inhibitor is also presented.

Description

Description: BACKGROUND OF THE INVENTION 1. Technical Field The present invention relates to recombinant DNA technology. In particular, the present invention relates to a cDNA gene sequence encoding a human placental ribonuclease inhibitor, a vector containing the gene, and a host containing the cDNA gene.

2. Background Art Through the development of recombinant DNA technology, DNA sequences from basically any organism can be easily cloned into plasmid or viral vectors for propagation in heterologous hosts. This shape can be used to study the sequence, structure, coding capacity or other properties of the DNA. Further, it can be used for various purposes such as detection of a complementary sequence in a sample, generation of a modified gene product, and regulation of biological functions by insertion into a new organism.

The advent of recombinant DNA (rDNA) technology has also enabled the isolation of coding sequences for complementary DNA (cDNA) using antibody probes.
Young, RA and RW Davis,
Proc. Natl. Acad. Sci. USA 80, 1194-1198 (1983). This system has the property of being able to best detect antigenic determinants (epitope) on polypeptides from DNA of any organism. Successful isolation of genomic or cDNA sequences using this system requires both a cDNA library and antibody probes of appropriate properties. Mierendorf, Robert C. et al., "Isolation of Genes by Screening of [Lambda] gt11 Library with Antibodies", Molecular Cloning Technology Guide (Guide
to Molecular Cloning Techiques), 152 , 458-469 (19
87). Using this technique, the gene encoding a particular protein is isolated and antibodies are raised. However, isolation of the gene using this technique is by no means guaranteed. For example, one may find that the sought construct kills a host cell, generally E. coli. In addition, when carbohydrates are added to a protein, particularly when synthesized in Escherichia coli, isolation of the gene may be difficult due to generation of antibodies mainly to the carbohydrate portion of the target protein. In such a case, the antibody cannot be used for detection. A further complication is the appearance of weak positives on the immunoscreening filter. The weak positives are proteins recognized by antibodies to contaminants in the immunoprotein preparation. This weak positive may be another protein that shares immunogenicity with the protein of interest.

Confidence that the target protein has been cloned depends on the activity of the recombinant protein synthesized in Escherichia coli, but many eukaryotic proteins are synthesized in Escherichia coli as incorrectly folded, insoluble and inactive forms. It is often impossible to be done. Hoess, A.
Et al., "Recovery of soluble, biologically active recombinant protein from whole bacterial lysate using ion exchange resin", Bi.
otechnology, 6, 1214-1217 (1988).

Inactive insoluble proteins may be solubilized and can be regenerated to an active form. However, determining the conditions required for efficient recovery of the active protein requires experiments to optimize many parameters that can affect the regeneration process. Marston, FAO "Purification of eukaryotic polypeptide expressed in E. coli", DNA cloning,
Volume III, Practical Methods, Chapter 4, DM Glover
r) Ed., IRL Press Edition, 1987 and references cited therein. Thus, testing a cloned gene encoding a particular protein depends on many factors that cannot be predicted until the experiment is started.

Generally, cDNA clones, that is, RNs for proteins
It is believed that the isolation of the double-stranded DNA corresponding to the information of A is primarily due to the sufficient purification of the protein of interest and the expression of antibodies to the protein in suitable animals such as rabbits. The next step is to use the antibody as an immunoprobe to obtain cDNA from a tissue in which such a protein is expressed.
Screening the library. A cDNA library is usually constructed with a bacteriophage lambda vector, especially with the bacteriophage vector lambda gt11 when antibody probes are available for screening. This is because lambda gt11 is an expression vector, and the fusion protein is E. coli beta-galactosidase,
It is meant to be formed between the native E. coli enzyme and the protein from the cDNA insert. Jend
risak), Jerry, et al. (1987) "[Lambda]
cloning of cDNA into gt10 and [lambda] tg11 ”,
"Guide to Molecular"
Cloning techniques), 152, 359-371 (1987). RNA D
The gene for the protein of interest present as an NA copy also exists under control of the E. coli promoter in lambda gt11.

When E. coli is infected with a recombinant bacteriophage encoding a protein of interest, some recombinant fusion protein is produced and released from the cells by lysis of the bacteriophage in plaques. This protein is picked up on a nitrocellulose filter, and the protein is detected with an antibody against the target protein. This screening operation is repeated until a uniform phage plaque having the foreign gene is obtained. The gene is then subcloned and expressed in a plasmid, a small circular DNA that can replicate independently of the DNA in the genome.

This method was applied to human placental ribonuclease inhibitor (PRI). Blackburn, Peter, et al., "Human placenta-derived ribonuclease inhibitors", The Journal of Biological Chem.
istry, 252 / 16,5904-5910 (1977). Blackburn (B
lackburn) disclosed a method for preparing soluble PRI. Here, it was purified 4000 times using a combination of ion exchange and affinity chromatography. PRI has a molecular weight of about 50,000
Was found to be an acidic protein.

Native PRI is a protein isolated from human placenta that specifically inhibits the enzyme ribonuclease (RNase), which catalyzes RNA degradation. It works by binding tightly to a wide range of RNases, and is very effective where strong RNase inhibition is required to protect RNA. PRI is a very promising protein for research and other uses. The physiological role of this protein is also unclear, but recent data indicate that the role of this protein in vivo may be in regulating vascular development. Shapiro, Robert and Ber
t) L. Vallee, "Human placental ribonuclease inhibitor inhibits both the angiogenin activity and the ribo- and creatase activities of angiogenin" Proc. Nat
I. Acad. Sci. USA, 84, 2238-2241 (1987). Shapiro (Sh
(apiro) and Vallee have revealed a relationship between PRI and angiogenin. Their experimental results indicate that PRI and related inhibitors are involved in the bioregulation of the vascular derived protein angiogenin from HT-29 human: adenocarcinoma cells. Human PRI has been shown to inhibit both the biological and enzymatic activities of angiogenin. Therefore, it was confirmed that the interaction of angiogenin / PRI was functionally significant. Tumor metastasis,
Potential interaction with PRI and inhibition of angiogenin by PRI are potential due to the involvement of angiogenin in creating pathological angiogenic conditions that occur in many diseases, including diabetic retinopathy and rheumatoid arthritis It could be an effective way to treat these diseases. PRI therefore has important mechanistic, physiological, pharmaceutical and / or therapeutic properties. Other features of the protein will also be discovered.

To study the role of the protein PRI in animals requires pure PRI, free of impurities present in preparations from human placental sources. These impurities are found in mammalian proteins, presumably prions and mammals.
DNA, some of which are derived from viruses such as HIV and hepatitis virus. Unfortunately, extracting a sufficient amount of purified protein from nature is relatively expensive. Therefore, there is a need for a large and inexpensive source of human PRI that is essentially free of impurities.

SUMMARY OF THE INVENTION According to the present invention, a useful amount of a human PRI clone-active gene product containing no impurities can be obtained. This procedure involves isolating the native gene encoding human PRI starting from a human PRI sample, screening a DNA library for cloned genes, forming a human PRI plasmid construct, introducing the plasmid construct into host cells and lysing cells. Includes the step of isolating human PRI protein by centrifugation.

The present invention also relates to a method for purifying, solubilizing and regenerating the isolated PRI, including the crude PRI obtained by the method described above.
Purification of human PRI by centrifugation, solubilization of human PRI by chemical reagents, and regeneration of human PRI by mixing solubilized PRI with a sufficient amount of buffer.

The present invention also provides a vector containing an inserted DNA gene sequence encoding human PRI, a host compatible with and containing a vector containing an inserted DNA gene sequence encoding human PRI, and a substantially pure set having a molecular weight of about 51,000 daltons. Replacement human PRI
Contains protein. This vector contains, for example, human
A plasmid DNA strand containing the foreign gene sequence encoding PRI is included.

The invention further includes a host cell, a promoter, and a recombinant host cell capable of expressing human PRI, including a foreign gene sequence encoding human PRI under the control of the promoter.

Further, in accordance with the present invention, the gene sequence encoding the recombinant human PRI has been determined.

Further objects, features and advantages of the present invention will become apparent from the following detailed description and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the plasmid pGEM Partial restriction of -7Zf (+)
3 shows a site and a function map.

 FIG. 2 shows the plasmid having the inserted PRI coding gene sequence.
Sumid pGEM Partial restriction site and function of -7Zf (+)
The map is shown.

Figures 3 to 3C show the nucleotide sequence of the cloned gene encoding human PRI.

FIG. 4 shows a partial restriction site and functional map of the pBR322 plasmid constructed with the inserted PRI-encoding gene sequence and expressed.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for obtaining a gene encoding human PRI, a method for expressing the gene product, and a method for purifying and regenerating recombinant human PRI. PRI is obtained from a host cell having a plasmid containing a gene encoding the PRI protein. The DNA sequence of the human PRI gene is also shown.

Important features of the invention include the isolation of inactive recombinant PRI, solubilization of recombinant PRI, and activation of solubilized recombinant PRI.

 Introduce a gene encoding recombinant PRI into host cells
A variety of vectors may be used for the purpose. To use vector
Is pGEM −7Zf (+), pBR322, PA3, pBC12B1, pGPD−
Various plasmids such as 2 and their derivatives and
Bacteria such as lambda gt11, T7 and their derivatives
Phages are included.

Typical host cells include both prokaryotes such as E. coli and eukaryotes such as yeast. A host cell in combination with a foreign gene sequence encoding PRI under the control of the vector and its promoter forms a recombinant host cell capable of expressing human PRI.

 Recombinant PRI is characterized by several properties.

a) molecular weight: 51,000 daltons b) inhibitory type: non-competitive In addition, the recombinant PRI of the present invention has several features to
Distinguished from RI. For example, unlike natural PRI, recombinant P
The N-terminal amino acid of RI is specified. Furthermore, unlike natural PRI, recombinant PRI is not inhibited and is not acetylated. Furthermore, the gene encoding the recombinant PRI does not include potentially harmful substances such as mammalian DNA, prions and HIV.

Inactive recombinant PRI exists as an inclusion body in the host cell. Generally, inclusion bodies are tightly packed, granular proteins. To isolate PRI from a host cell, its cell membrane must be disrupted. Several cell lysis techniques may be used, but the preferred method is to use an appropriate amount of lysozyme followed by centrifugation and sodium dodecyl sulfate (SDS)
Secondary processing is included. In this way about 0.
1% soluble active PRI and 99.9% insoluble inactive PRI result. The insoluble protein is removed as a crude inert protein by centrifugation. This protein is further purified by a buffer suspension / centrifugation method.

After isolating and purifying the inactive PRI protein, it was solubilized, ie put into solution. Solubilization is first preferably initiated in a buffer solution containing chemical reagents that cause the polynucleotide chains to separate from one another in solution. Useful chemical reagents include urea, guanidine hydrochloride and SDS, but urine is preferred. Prior to use, the urea solution is preferably centrifuged to remove unwanted cell lysates. This solubilization step should be performed in a relatively short time, ie at most 30 minutes. It was found that there was an inverse relationship between the time of combining the inactive PRI with the chemical reagent and the final yield of active PRI.

The activation step involves diluting the PRI containing solution with a buffer solution and maintaining it for a time sufficient to regenerate the active state. Several factors are important for the activation state. For example, the dilution step is relatively quick, ie, 2
It should be done within minutes, preferably within 30 seconds. The pH of the buffer solution must be in the range of about 6.5 to about 8.5, and is preferably about 7.5. It is also effective to include an active agent such as glycerin or sucrose in the buffer solution. It has been discovered that the presence of a sufficient amount of activator in the buffer solution can increase the ability of the PRI protein to regenerate. It was also found that a dilution ratio of about 1 part of the PRI solution to about 100 parts of the buffer solution was optimal for assisting the PRI regeneration.

At a temperature of about 10 ° C. to 25 ° C. for at least 8 hours after dilution of the solution,
Preferably, it must be allowed to stand for 8 to 18 hours. Under these conditions, the PRI protein is regenerated and about 7% of the PRI molecules become active.

The following examples are provided to illustrate how to generate cloned genes encoding human PRI and how to generate active recombinant human PRI.

Example 1 Isolation of Lambda gt11 Clones Containing the Human PRI Gene Before cloning the human PRI gene, substantially pure native human PRI protein must be obtained. This human PRI protein was prepared by the method of Blackburn, which is incorporated herein by reference for the description of the purification method.
urn) etc. (described above). Briefly, soluble ribonuclease inhibitors from human placenta can be purified 4,000-fold by a combination of ion exchange and affinity chromatography as described by Blackburn et al. (Supra).

PRI protein from natural sources was purified to apparent homogeneity by the method described by Blackburn et al. (Supra). This protein is also DEAE Sepharose CL
Purification by binding to -6B and gradient elution removes functional contaminants that are not apparent in gel analysis.

An experimental animal New Zealand white rabbit was immunized with 1 mg of purified PRI protein, followed by a second immunization.
They were bred for 0 weeks. A commercial cDNA library of human placenta in bacteriophage lambda gt11 (Clontech, Palo Alto, Calif.) Is listed in the Protoblot Immunoscreening Manual (Promega, 1987), which is incorporated herein by reference. Screened according to the method. Briefly, this library was used at a density of 50,000 plaques per plate for E. coli Y1090 (r
Minus) were plated on the strain and the 10 plates were screened for a total of 500,000 plaques. Plaques showing strong positivity were identified.

A nitrocellulose filter pre-soaked in isopropyl-beta-D-thiogalactopyranoside (IPIG) was placed on this plate. Following adsorption of the protein to the filter, the filter was lifted and the remainder of the protein binding site on the filter was blocked by incubation in a buffer containing 1% v / v bovine serum albumin. The filter was then immersed in a 1000-fold dilution of the rabbit antibody in buffer and shaken warm at room temperature for about 30 minutes. The filters were washed three times with buffer and soaked in a 1: 7500 dilution of a commercially available goat anti-rabbit IgG alkaline phosphatase conjugate (Promega). This "secondary antibody" detects the presence of the primary antibody bound to the PRI fusion protein on the filter. Room temperature in the presence of secondary antibody 30
After a one minute incubation, the filters were washed three times with buffer. A substrate solution of alkaline phosphatase was added to develop color. 50,000 plaques showing strong positivity
Observed from screening plaques. Strongly positive plaques were purified to homogeneity by excising and plating plaques until the specific phage was pure.

DNA derived from recombinant lambda phage having a putative PRI gene was purified by an immunoprecipitation method using a commercially available phage absorbent (Lambdasorb, Promega).
Restriction analysis of phage DNA indicated the presence of a single 1.6 kilobase (kb) insert. This insert could be removed from the phage DNA by digestion with the enzyme EcoRI. This insert is sized to carry the entire PRI gene, since the PRI protein requires a 14 bp gene to encode it. In general, eukaryotic messenger RNAs are longer than required for protein coding sequences, due to the increase in 5 'untranslated, 3' untranslated and polyA regions.

Example 2 Subcloning of a gene containing a PRI insert into a plasmid
In Example 1, the PRI gene was combined with a single EcoRI fragment.
I took it out of Lambda GT11. Therefore this fragment
Plasmid directly into plasmid pGEM -7Zf (+) (Promega
Sub-cloning. FIG.
Rasmid pGEM -7Zf (+) was shown. This fragment
PGEM Subcloning into -7Zf (+) plasmid
The expression of the correct reading frame for this insert.
Came.

 pGEM At the EcoRI site of the -7Zf (+) plasmid
The reading frame is for the EcoRI site of bacteriophage lambda gt11.
Same as the ones. Therefore, if the cDNA coding sequence
Is expressed in lambda gt11 if pGEM -7Zf (+)
It will also be expressed in Rasmid.

 Insert this fragment into the vector in the correct orientation
The promoter on this plasmid, ie 1a
The c promoter drives the expression of the PRI insert. PRI-tan
It is the first of beta-galactosidase attached to the protein
Several amino acid residues of pGEM In the -7Zf (+) plasmid
Before ATC for multiple cloning regions and PRI proteins
Encoded by a 5 'non-coding region in an insert
Extra amino acids.

 Bacteriophage lambda g with EcoR I PRI insert
t11 is digested with EcoR I, Maniatis, etc.
(Molecular Cloning, Laboratory Manual, (198
2), Cold Spring Harbor, Laboratory Pre
Edition, Cold Spring Harbor, New York)
PGEM according to standard cloning method -7Zf (+) plasmy
Subcloned into The location of this recombinant plasmid
FIG. 2 is shown as a diagram.

Example 3 pGEM Expression of PRI insert in -7Zf (+) plasmid When transferred to the -7Zf (+) plasmid,
The input is in the open reading frame for expression. Ie this plus
Expression reading frame at the EcoR I site of the mid is in lambda gt11
It is the same as the expression reading frame of the EcoRI site. Therefore, this
PRI fusion proteins can be produced directly in E. coli. pGE
M For induction of 1ac promoter of -7Zf (+) plasmid
About 60,000 daltons following induction of expression
Natural PR using a fusion protein with molecular weight as a probe
Western blot using primary rabbit antibody to I
Analysis (Towbin, H, et al., Proc. Natl. Acad. Sci.
U.S.A. 76, 4350 (1979)).

The fusion protein produced is expected to be somewhat larger than the native PRI. In fact, this was again observed, consistent with the assumption that the entire coding region of PRI is present in this EcoRI insert.

Example 4 Production of PRI protein in rabbit reticulocyte lysate pGEM of PRI insert Cloning into -7Zf (+)
Later, this vector uses the SP6 promoter on the vector
Can be used to synthesize RNA from in vitro inserts
It is. RNA is synthesized from this promoter and
Used for reticulocyte lysate programs. Natural PR
51,000 dalton protein with the size of an I protein
The quality was synthesized. Again this result shows that all PRI
Indicates that it is contained on the clone.

Example 5 pGEM Sequence of the insert in the -7Zf (+) plasmid
The first sheet at the 5 'and 3' ends of the PRI gene insert
Kenning is pGEM Clones in the -7Zf (+) plasmid
I went there. This sequencing data is
The presence of the ATG codon a few nucleotides downstream of the
And 36 residues at the 3 'end of the cloned gene
The presence of a group polyA tail was indicated. According to Example 4
Due to the size of the fusion protein produced, PRI protein
The logical starting point of a quality coding sequence is a sequencing clone
Can be concluded to be the first ATG codon found in
You.

 Further sequencing data is available from a commercially available deletion system
(Erase-a Base ) System
PGEM using Promega -7Zf (+) clone
Obtained by successive exonuclease III deletions.
Exonuclease III is D from the 5 'protruding end or blunt end
Specific digestion of NA, while leaving 3 'overhanging 4 bases
Used to do. The constant digestion rate of this enzyme
By removing a partial sample from the reaction
Loss can occur. Substantially cloned inserts with this tactic
The entire nucleotide sequence of the product was generated. Like this
The nucleotide sequence of the cloned human PRI gene
It is shown in Fig. 3-3C. Here is the derived protein
Amino acid sequence was included.

Given the nucleotide sequence of the cloned gene for human PRI, it is within the scope of the present invention to construct a hybridization probe that allows isolation of the PRI gene by hybridization of a cDNA library.

Example 6 Generation of a Gene Expressing a Native Sequence Protein To synthesize a native sequence protein without a fusion moiety linked to the gene product, the first ATG codon found in the sequencing of the clone is the natural translation initiation Codons were assumed. A prokaryotic ribosome binding site was inserted 6-8 bases from the ATG start site to allow the start site to function in E. coli. Data from other constructs show that a PRI chimeric protein with eight extra amino-terminal amino acids is lethal when expressed from a regulatable E. coli promoter. So Sudasia
r) F. William and Barbar
a) A. Moffatt ("Bacteriophage T7RN for selective expression of cloned genes"
Use of A polymerase ", J. Mol. Biol. 189, 113-130 (19
86)) PRI with T7 promoter as described
I decided to induce the gene.

After completion of this construct, it was amplified in an E. coli host lacking T7 RNA polymerase. So this gene is
Not expressed until transferred to host expressing RNA polymerase. Such expression systems have been proposed (see Studier et al., Supra, and Rosenberg, Alan, H. et al., "Vectors for the selective expression of cloned DNA by T7 RNA polymerase". "Gene, 56, 125-135 (1987)) Also basically performed in this chapter.

In a preferred construct, the ribosome binding site and the region before the ATG codon and the untranslated leader sequence following the Shine-Dalgarno sequence are the bacteriophage T7 gene.
10, derived from leader and ribosome binding sites. In addition, a T7 promoter is present to produce this RNA.

 PGEM of PRI gene from lambda gt11 -7Zf (+) plastic
The transfer to the sumid is based on the fact that this gene
Wide because it is adjacent to the cloning restriction enzyme site
Cloning tactics.

 Cut the PRI gene as a BamHI-AatII fragment
Between these two sites in the export plasmid vector pBR322.
Put in. Rosenberg et al. (Above)
As shown and synthesized as a synthetic oligonucleotide.
The T7 terminator of the sequence
And between the Aat II sites. Xba I site is BamH I
-PGEM during transfer of the PRI gene of the Aat II fragment
It is derived from a -7Zf (+) linker.

Synthetic nucleotides are T7 promoter and gene 10
Those containing the 5 'untranslated sequence and the gene 10 Shine-Dalgarno sequence were synthesized.

This oligonucleotide was cloned into the BamH I and Bs
Inserted at the beginning of the PRI gene between the tXI sites. Initial sequence analysis of the PRI insert revealed that a BstXI site was present after the third codon of the putative gene sequence. BstXI was designed to replace these amino acids because it cuts out the coding sequence. The final construct is shown in FIG. It contains the PRI gene in plasmid pBR322, which was made to be expressed in E. coli.

In order to express the PRI gene in this synthetic construct, the plasmid shown in FIG. 4 was placed in an E. coli strain producing T7 RNA polymerase. This strain was constructed according to the methods of Studier et al. (Supra) and Rosenburg et al. (Supra). E. coli JM109 strain was lysogenized with wild-type phage lambda. This strain was used as a host for infection with recombinant lambda phage (lambda D69-T7) carrying the gene for T7 RNA polymerase inserted into the BamHI site of the lambda vector. The T7 RNA polymerase gene was inserted into the chromosome of E. coli JM109-DE3. This clone was registered on February 21, 1990 with the American Type Culture Collection (12850 Parklone Drive, Rockville, MD 20850) under accession number 68230.

The culture was grown to an optical density of 1 and induced with IPTG. The PRI protein produced was monitored by polyacrylamide gel electrophoresis (PAGE) of bacterial proteins (Laemmli, UKNature (London), 227,680).
(1970), followed by Western blotting (Towbin, H. et al., Supra) and detection with a primary rabbit antibody to the PRI protein. IP in this analysis
Induction of PRI culture by TG and culture after 4 hours culture 1
It was shown that about 40 mg of PRI was synthesized per liter.

Example 7 Production of active PRI in E. coli JM109 E. coli JM109- harboring the PRI plasmid described in Example 6
DE3 can produce certain levels of active PRI protein.
Although most proteins are synthesized in an inactive and insoluble form, about 0.1% of all PRI proteins are soluble and active. Active PRI protein bound RNase
Purified from centrifuged French-pressed lysates using batch adsorption on an affinity matrix containing (Scopes, Robert K. "Principles and Practices of Protein Purification", 2nd ed., 27, 1987). Collected in this way
PRI has the same molecular weight as gels as the non-glycosylated proteins reported by native PRI, Blackburn et al. (Above) and Blackburn (above), and has almost the same specific activity as purified natural products. Have.

Since most of the PRI protein produced in E. coli is synthesized in an insoluble form, the insoluble material must first be solubilized and regenerated to the active form in order to find an economical way to produce active recombinant PRI protein.

Example 8 Production of PRI Protein Under the Control of the Escherichia coli Tac Promoter Under the Control of the Strong Host Promoter pTac in the pTTQ Vector (Stark, JJR, Gene, 51, pp 255-267,19
87) Natural PRI protein was produced in E. coli. PRI
The insert was removed from pBR322 as a SphI-EcoRI fragment. This fragment contains an excellent ribosome binding site in front of the PRI gene, and the vector pTTQ19
(Amersham) was cloned between the Sph I and EcoR I sites. This construct is designed to express the PRI gene with the Tac promoter on the bicistronic message. One ribosome binding site is from the pTTQ vector and the other is from the PRI insert. Since the internal ribosome binding site works well in E. coli, the native PRI protein, but not the fusion protein, is synthesized within this construct. This construct was placed in several different E. coli strains to find a host that gave the highest levels of soluble and active protein. Hosts tested included E. coli C6000, JM109, NM522, BSJ72, B121 and
HB101 is included. At least some soluble and active PRI was produced in each host tested.

Example 9 Secretion of the PRI protein in E. coli The PRI protein will have many disulfide bonds since it has 32 cysteines per molecule by amino acid analysis of the native protein. These bonds are likely not formed correctly in the reducing environment of the cytoplasm. Therefore, it was designed to secrete this protein into the periplasmic space of bacteria so that disulfide bonds occur under more oxidative conditions. Synthetic oligonucleotides encoding the signal sequence of the E. coli ompA protein in front of the start of the PRI gene (Movva, NR, J. Mol. Bio
l.143pp.317-238, 1980) to give pBR322
-Fixed PRI. These leader amino acids are for secreting the protein, and are cleaved by E. coli signal peptidase when the protein is secreted from the cell. In this construct the T7 promoter is
Continue to work for transcription of the PRI gene as in pBR322-PRI, but the ompA signal sequence is created first. The results showed that PRI was correctly processed (removal of the signal sequence) and secreted from the cells. The secreted PRI protein was found to be inactive. In addition, protein levels were about one-tenth that produced intracellularly and only about 4 mg / liter of culture.

Example 10 Example of PRI construct lethal to E. coli
PR by letting these constructs act lethal to host cells
There are several conditions in which the expression of
Was. One is the 12 extra amino-terminal amino acids in the vector pTTQ9.
This is the case when an attempt is made to construct a PRI fusion having an amino acid.
The other is a secretion expression vector PIN NIII ompA (Masu
i), Y. et al., “Multipurpose expression cloning vector in E. coli.
Hicle "," Experimental manipulation of gene expression ", M. Inoue (In
ouye) edition, Academic Press Edition, 1983)
This is the case now. In the former case, first cut pTTQ9 with Pst I
After blunting the ends with Klenow, the vector is
Ligation. pGEM -7Zf (+)-PRI insertion of PRI
The input was removed as a BamHI-SphI fragment and the Pst
 BamHI and EcoR of pTTQ9 vector with blunted I site
Cloned between the I sites. By Sal I and Kpn I
Cleavage of these cohesive ends and Klenow treatment and ligation
From the strong Tac promoter on the vector
PRI genetics when matching the open reading frame for protein expression
The child could be located. But the final construct is obtained
This expression system was not
Concludes that it produces a lethal dose of the PRI fusion protein
It is. Later, the same fusion protein is basically
Control of the T7 promoter in the same construct as pBR322-PRI
Under control, but in this case an extra
Using clones made to incorporate 12 amino acids
Was shown. Fusion dandelion in expression system
The difference in quality of production is probably due to
It may be due to stronger suppression of the system.

 Cloning of the PRI insert into the secretion vector pIN III ompA
Lethality was also observed in the transformation. Insert this PRI insert into BstX1
-PGEM as EcoR1 fragment -7Zf (+)-PRI or
EcoR1 cleaved and phosphorylated pIN III ompA
Cloned. This PRI fragment has two directions
Inserted, resulting in P
The RI gene is located before and after. But the only view
The construct to be observed is that the PRI insert is inserted after this promoter.
And therefore, does this gene expression occur?
Was. Introductory constructs are lethal for the reasons mentioned earlier
It seems.

Example 11 Isolation and recovery of insoluble PRI produced in E. coli Insoluble PRI produced in E. coli induced to produce PRI can be recovered as follows.

1. The cells induced to produce insoluble PRI are suspended in a buffer. Suitable buffers are 50 mM Tris HCl (pH 7.5), 5 mM EDTA
And a TESDTT buffer containing 5MMDTT. Suitably, the suspension generally contains about 20 grams of frozen cells in 400 ml of buffer solution. Changing the scale of this process is within the scope of the present invention. However, it is preferred to maintain a ratio of 1:20 (grams of cells / ml of buffer volume).

2. Stir the suspension vigorously in ice for about 10 minutes.

3. Destroy cell membrane by cell lysis. A cell lysis method in which powdered lysozyme is added at a concentration of 1 mg / ml (Sigma, class VI) and stirred in ice for 30 minutes is preferred. After stirring, about 4 ml of 10% (v / v)
Add sodium dodecyl sulfate and stir again in ice for 30 minutes.

4. Apply this suspension at about 4 ° C for about 20 minutes, about 17,000x
Centrifuge at g. A two-phase pellet consisting of a gray solid lower layer and a translucent, highly viscous layer and supernatant is obtained.

5. Carefully decant the supernatant from the pellet, leaving only the pellet. This pellet is the crude PRI protein.

Example 12 Purification of PRI 1. Resuspend the crude PRI pellet obtained in Example 11 in the buffer under the conditions of Example 11.

2. Apply this suspension at about 4 ° C for about 20 minutes and at 17000
Centrifuge at xg. After centrifugation, a highly viscous layer is formed.

3. Decant the supernatant from the centrifugation mixture.

4. Resuspend the pellet in buffer with stirring. Preferably, this buffer solution is 400 ml of TE5DTT per 20 grams of pellet. The suspension is stirred vigorously in ice for about 30 minutes.

5. Apply this suspension at about 4 ° C for about 20 minutes, about 17,0
Centrifuge at 00 × g. The remaining pellets are light gray.

Example 13 Solubilization of PRI The pellet was suspended and solubilized as follows.

1. Suspend the final pellet in preferably 200 ml of TE5DTT buffer and stir in ice for about 120 minutes. A milky suspension is obtained.

2. Solubilization preferably starts in a buffer solution containing urea at a concentration of about 4M. TE5DTT is suitable as a buffer solution. The urea solution is 800ml TE5DTT containing 360g urea
It is desirable that

3. Solubilized PRI is rapidly stirred at about 10 ℃ to 25 ℃
Dilute at a temperature of about 200 ml to 800 ml of buffer solution. Stirring this solution for about 3-5 minutes results in a nearly clear solution containing some cell debris.

4. Before use This urea solution is preferably centrifuged to remove insolubles. Centrifugation is performed at 6000 × g at about 4 ° C. for about 15 minutes. The supernatant becomes colorless and transparent. This supernatant contains the solubilized PRI.

Example 14 Activation of PRI This supernatant is quickly diluted in 100 buffer solutions and maintained for a sufficient time to allow PRI to regenerate to an active state. This buffer solution is preferably TE5DTT containing 10% (v / v) of a water activator. Sucrose can be used, but glycerin is preferred as the water activator. The pH of the buffer solution is about 6.
It is in the range of 5 to about 8.5, preferably 7.5.

After leaving the solution at room temperature for about 8-18 hours, the PRI protein regenerates and about 7% of the molecule becomes active.

Example 15 Separation of Regenerated and Inactive PRI Regenerated PRI in 100 is diethylaminoethyl (DEAE)
It can be concentrated in a ram reactor. DEAE column uses recycled PRI
Not only concentrates but also aids in the PRI protein regeneration process
This is an advantage of the present invention.
I have. Use a typical column at a flow rate of 30 liters / hour.
Cuno ) There is a 3200DEAE cartridge. less than
Indicates an operation.

1.Reproduced PRI is sent to the cartridge by pump, and 0.5M Na
Elution is performed with TE5DTT containing Cl.

2. The eluate from the column was passed through an affinity resin containing RNase covalently bound at a concentration of at least 1 mg / ml. Active P
RI adsorbs to RNase, while inactive and illegally regenerated material escapes through the affinity column.

3. Elute active PRI from the affinity resin with a 0.05 M sodium acetate buffer (pH 5.0) + 3 M NaCl solution containing 5 mM DTT. At this stage the PRI is basically pure, PAGE
And uniform.

4. If RNase contamination is a problem, binding to DEAE Sepharose CL-6B and concentration gradient elution can further remove functional impurities from the PRI protein. The yield of PRI from 20 grams of cells due to the regeneration and purification steps is about 3 million units.

Recombinant proteins can be distinguished from native proteins by the presence of their unprotected amino termini. Native PRI cannot be sequenced from its amino terminus, probably because its amino terminus is protected by acetylation. Protein sequencing of the recombinant protein revealed the presence of the unprotected amino terminus of the recombinant, further indicating that the N-terminal methionine was removed in E. coli.

It is to be understood that the invention is not limited to the specific forms shown herein, but rather encompasses modifications within the scope of the following claims.

────────────────────────────────────────────────── ─── Continued on the front page (58) Field surveyed (Int. Cl. ⁷ , DB name) C12N 15/00 C12N 1/21 C12P 21/02 BIOSIS (DIALOG) WPI (DIALOG)

Claims (5)

(57) [Claims] 1. A plasmid containing a DNA sequence encoding a human placental ribonuclease inhibitor in the same DNA segment as the segment of the plasmid pBR322-PRI, wherein the DNA sequence is between the disrupted BamHI and disrupted AatII sites shown below. A plasmid comprising a DNA sequence corresponding to a sequence encoding a human placental ribonuclease inhibitor present in the plasmid. 2. The plasmid according to claim 1, which is a plasmid pBR322-PRI. 3. An Escherichia coli strain containing an inducible T7 RNA polymerase gene and transformed with the plasmid of claim 1 or 2. 4. The E. coli strain according to claim 3, which is a transformed JM109-DE3 strain. 5. A method for obtaining a cloned active human placental ribonuclease inhibitor, comprising the steps of:
Culturing the Escherichia coli strain described in 1 above, and isolating the active form human placental ribonuclease inhibitor thus produced.